Slight bone damage, such as a fracture, or the like, may be cured by simple surgical treatment, but there are bone diseases such as nonunion, delayed union, bone loss, bone necrosis, or the like, for which it is difficult to find alternatives except for autogenous bone graft since access to osteoblast cells is fundamentally difficult, or the number of cells or activity thereof are insufficient. A treatment method using osteoblast cells cultured in vitro is recognized as a new alternatives to solve this difficulty. The osteoblast cells are derived from mesenchymal stem cells and may be partially obtained from normal bone, fat, blood, or the like, but a harvest site at which minimally invasive harvest and the maximum yield of cells may be expected is bone marrow. In the bone marrow, a large amount of bone marrow stromal cells referred to as the mesenchymal stem cells are present as well as blood-related cells. A technology of regenerating bone using the bone marrow stromal cells may be largely classified into 1) a technology of using bone marrow itself, 2) a technology of separating only nucleated cells from the bone marrow to use them, 3) a technology of proliferating the bone marrow stromal cells to use them as a cell therapy product, 4) a technology of proliferating and differentiating the bone marrow stromal cells into osteoblast cells to use them as a cell therapy product, and the like. Some of them were developed, and some of them have been currently developed.
In order to induce bone regeneration using the cells as described above, a growth factor capable of stimulating and promoting signal transduction pathways for bone differentiation has been demanded. In addition, in osteoarthritis, rheumatoid arthritis, a bioactive material capable of curing damaged cartilage and regenerating bone tissues at the same time in addition to controlling inflammation has been demanded. Through many studies, it was known that human recombinant bone morphogenetic proteins (rhBMPs) forms new bone and new cartilage as a TGF-β super-family when rhBMPs is grafted ((Wozney J M et al., Science 1988; 242:1528-1534; Celeste A J et al., Proc. Natl. Acad. Sci. US 1990; 87:9843-9847). Currently, 20 or more BMPs were discovered, and among them, rhBMP-2, -4, -5, -6, -7 (Kim C S et al., J Periodontal 2002; 73:1126-1132; Wikesjo U M et al., J Clin Periodontal 1999; 26:392-400; Kingsley D M et al., Cell 1992; 71:399-410), and the like, have excellent osteoinductive ability to thereby be used in a bone tissue engineering field. Nevertheless, at the time of applying the BMP to patients wanting the bone regeneration therapy, the BMP should be used at a significantly high concentration ((1.5 mg/ml) and a cost thereof is expensive, such that it is difficult to clinically apply the BMP. However, an innovative protein capable of replacing the BMP was found in 1998, and it was reported that the protein, which is referred to as intercellular LIM Mineralization Protein (LMP)1 acting in cells, is directly involved with differentiation of osteoblast (Boden S D et al., Endocrinology 1998; 139:5125-34). This protein has sites capable of binding with two WW domains in an osteoinductive region, wherein the portion binds to the WW domain of Smad ubiquitin regulatory factor (Smurf)1 decomposing Smad1 and Smad5 that are the main proteins for signal transduction pathways involved in bone differentiation. In the study reported in 2006, it was confirmed that when affinity of the LMP1 to Smurf1 are compared with those of Smad1, and Smad5, the affinity of Smurf1-LMP1 are higher than those of Smurf1-Smad1 or Smurf1-Smad5, and the shortest sequence capable of inducing differentiation into the osteoblast cell and bone formation in a WW domain binging site of LMP1 was identified (GAPPPADSAP, Boden S D et al., JBC 2006; 281:17212-17219).
However, in order to synthesize the domain having strong hydrophobicity in vitro to induce the bone regeneration, the domain has an activity only when the domain is permeated into cells in which smurf1 is present. As a result of the studies for solving this problem, a protein transduction domain (PTD) was suggested. Recently, studies of the protein transduction domain (PTD) capable of effectively transporting a protein which it is difficult to permeate through a cell membrane into the cell have been actively conducted. It is known that this protein transduction domain (PTD), which is a short peptide with positive charge, may permeate through cell membranes and effectively transport DNA, RNA, fat, carbohydrates, compounds, or virus into cells, in addition to protein. The principle that the PTD permeates through the cell membrane is not found yet, but is thought to be independent on a receptor, endocytosis, or phagocytosis. This cell membrane permeation phenomenon of the peptide shows that movement of therapeutic proteins, which is difficult to be used as a drug due to a short half-life in vivo, and movement of genes, which are macromolecules, may be improved, such that pharmaceutical effects thereof may be improved.
Among them, studies of TAT protein, which is a human immunodeficiency virus-1 (HIV-1) transcription factor have been mainly conducted. It was found that the TAT protein is more effective in passing through the cell membrane, when the protein consists of some of 47th to 57th amino acid sequence (YGRKKRRQRRR) on which positive charged amino acid are intensively distributed, as compared to when the protein has a complete form consisting of 86 amino acids (Fawell, S et al., Proc. Natl. Acad. Sci. USA, 91:664, 1994). It was reported that TAT PTD may permeate alone or be attached to another protein to permeate through the cell membrane, and transport protein of 10 to 120 KDa regardless of the receptors into cell (Schwarze S R. et al., Trends Pharmacol Sci 2000; 21:45-48; Fawell S et al., Proc. Natl. Acad. Sci. USA 1994; 91:664-668).
As another example of peptides of which effects as cell penetrating peptide (CPP) are confirmed, there are peptides having an amino acid sequence from 267th to 300th amino acid of herpes simplex virus type 1 (HSV-1) VP22 protein (Elliott, G. et al., Cell, 88:223, 1997), peptides having an amino acid sequence from 84th to 92th amino acid of HSV-2 UL-56 protein (GeneBank code:D1047[gi:221784]), peptides having an amino acid sequence of 339th to 355th amino acid of antennapedia (ANTP) protein of Drosophila sp. (Schwarze, S. R. et al., Trends. Pharmacol. Sci., 21:45, 2000), and the like. In addition, effects of artificial peptides consisting of positively charged amino acids were also confirmed (Laus, R. et al., Nature Biotechnol., 18:1269, 2000).
Since it was found that in the case in which the CPP according to the related art is connected to another peptide or protein, the CPP effectively transport the fused protein into cells, various applications using the CPP were tried (Korean Patent Registration No. 10-0568457). However, since the cell permeable peptide is derived from a virus, there is a problem in view of safety.
Therefore, the technology development for safe peptides capable of regenerating bone and treating diseases including inflammatory diseases through optimal target orientation, instead of the existing virus derived peptide receptor has been urgently demanded, but research achievements are still insufficient.
Therefore, the present inventors have studied in order to synthesize a safe cell permeable peptide capable of treating diseases including bone diseases, inflammatory diseases, and excellent target specificity. As a result, the present inventors newly synthesized human bone morphogenic protein-4 derived heparin binding domain (HBD; SSRKKNPNCRRH) containing a large amount of cationic amino acids such as arginine, and the like, as a cell permeable domain, identified that impermeable domains may be effectively transported into cells using this cell permeable peptide, and confirmed that intracellular transport of a target active domain and expression of a target gene may be significantly increased by using this peptide as the peptide for transporting the active domain into the cell, thereby completing the present invention.